Importance of detecting specific targets. Methods for detecting specific molecular, cellular, and viral targets are fundamental tools for medical and veterinary diagnostics, environmental testing, and industrial quality control. Examples of methods for detecting specific targets in clinical medicine include over-the-counter rapid pregnancy tests, microbiological culture tests for determining the resistance of infectious agents to specific antibiotics, and highly automated tests for cancer markers in blood samples. Detecting pathogen contaminants in food, high throughput screening of candidate compounds for drug discovery, and quantifying active ingredients in pharmaceuticals exemplify industrial manufacturing applications that depend on methods for determining the presence of specific targets. Environmental applications requiring testing for specific targets include detecting water supply contamination, airborne biothreat agents, and household fungal contaminants.
Desirable attributes of methods for detecting specific targets. Methods for detecting specific targets should be accurate, that is they should be sensitive and specific. The methods should be sensitive enough to detect the target when it is present in significant amounts. And they should be specific; they should not indicate the presence of the target when it is not present in significant amounts. Other beneficial attributes include breadth of potential target analytes, rapid results, ease-of-use, cost-effectiveness, target quantification, and automation. The importance of the various desirable attributes can depend on the particular application and testing venue.
Breadth of targets. Testing methods should be capable of detecting a wide range of specific targets. Representative target classes include human cells (e.g., CD4+ cells in HIV/AIDS diagnostics), bacterial cells (e.g., Methicillin Resistant Staphylococcus Aureus or MRSA or E. coli), viruses (e.g., Hepatitis C virus), prions (e.g., Bovine spongiform encephalopathy agent, the cause of “mad cow” disease), macromolecules (e.g., proteins, DNA, RNA, carbohydrates), and small molecules (e.g., chemotherapeutic drugs, lipids, sugars, amino acids, nucleotides),
Labeling specific targets. One important approach for detecting specific cells, viruses, or molecules is to tag the targets with optically detectable labels that bind specifically to the target. Target-specific labels can have various types of target binding moieties including macromolecules (e.g., antibodies, protein receptors, nucleic acids, carbohydrates, and lectins) and small molecules (e.g., hormones, drugs of abuse, metabolites). The detectable signaling moieties of the target-specific labels can use a variety of signaling modes including fluorescence, phosphorescence, chromogenicity, chemiluminescence, light-scattering, and Raman scattering.
Methods for determining the presence of specifically labeled targets. A variety of methodologies have been developed for testing samples for the presence of specifically labeled target molecules, cells, and viruses. The technologies differ in the types of samples accommodated, mode of binding of the label to the target, type of signal delivered by the label, plurality of labeled targets detected, method for distinguishing bound from unbound labels, and technology for detecting the specifically labeled targets.
Which method is used depends on the type of analyte to be detected, testing venue, degree of automation required, clinically relevant concentration range, skill of the operator, and price sensitivity. For example immunoassays use target-specific antibodies for detection. To make them optically detectable, antibodies can be attached to signaling moieties including radioactive isotopes, fluorescent molecules, chemiluminescent molecules, enzymes producing colored products, particles dyed with fluorescent compounds, resonance light scattering particles, or quantum dots. Antibody-based technologies include manual lateral flow, ELISA, flow cytometry, direct fluorescence immunoassays, western blots, and highly automated central laboratory methods. Similarly, detection of specific nucleic acid sequences can use complementary nucleic acid probes associated with a variety of types of signal moieties using methods that include nucleic acid amplification, Southern and Northern blots, and in situ hybridization.
Using imaging to count labeled targets. Imaging is a powerful method for detecting specifically selected labeled targets on a detection surface. Imaging methods map the optical signal emanating from each point in the detection area to a corresponding point in the image. In contrast, non-imaging detection methods generally integrate the optical signal emanating from the entire detection area.
Some imaging methods can detect and count individual labeled target molecules. Enumerating specifically labeled target molecules can result in detection at very low target levels compared to detection area integration methods. The sensitivity advantage of imaged-based target counting methods stems chiefly from the fact that the optical signal to background stays essentially constant as target levels decrease. In contrast, for detection area integration methods the signal to background decreases as the target levels decrease. One type of method builds an image by systematically scanning the detection area with a microscopic beam. Scanning methods are more time consuming than methods that use digital array detectors (e.g., CCD or CMOS cameras) to simultaneously enumerate specifically labeled targets in the entire detection area.
Large area imaging at low magnification for sensitive target counting. Some methods use high magnification microscopy to enumerate the individual microscopic targets. Microscopic imaging lacks sensitivity because each image only samples a small area. Larger areas can be successively imaged but acquisition of many images can be laborious, expensive and time consuming. Alternatively, labeled microscopic targets can be individually detected and enumerated using large area imaging at low magnification. Low magnification imaging can enumerate a small number of microscopic targets in a relatively large area in a single image.
Some methods that use large area automated digital imaging have been developed for simultaneously detecting individual labeled targets. These methods generally detect labeled targets in a capillary chamber and use lateral flow to remove unbound label. As for other lateral flow methods, this technical approach complicates automation and limits the volume of sample that can be conveniently analyzed.
Using selection to isolate specifically labeled target from free label and other labeled entities. To detect specifically labeled targets methods generally must remove or distinguish unbound label and other labeled entities from the bound label. One common approach uses physical selection of the labeled target complexes after which the free labeled can be removed by repeated washing. This approach, while effective, generally requires labor for manual methods or sophisticated liquid handling engineering for automated systems.
Selection of labeled target complexes can be mediated by physical capture on a surface coated with a binding moiety specific for the target (e.g., capture antibodies in ELISA or lateral flow assays or DNA probes in microarray assays). Similarly, separating labeled target complexes from free label can be done by using selection particles coated with target-binding moieties. For example, magnetic selection can be used to select labeled target complexes bound to target-specific magnetic particles during wash steps that remove free label.
Lateral flow methods can simplify the washing process to some extent by using capillary flow to wash free label from labeled targets that are selected by capture on a surface. This type of washing may require an addition of a wash solution as an extra user step. Lateral flow methods are not very sensitive and not generally amenable image analysis, automation, or high throughput.
Methods that do not require washing to remove free label from specifically labeled targets. Several methods have been developed that detect targets specifically complexed with labeled target-specific binding moieties. One type of method uses labels that do not emit signal unless they are bound to the target molecules. These labels have the limitation that they do not emit a strong enough signal for efficient large area detection of individual labeled targets. Another method that does not require washes uses selection through a liquid phase barrier to separate labeled target complexes from unbound label. This approach uses detection area integration rather than sensitive image analysis and thus lacks high sensitivity.